溃疡性结肠炎（UC）是直肠和结肠的非特异性炎症性疾病，与克罗恩病（CD）同是典型的炎症性肠病。UC具有难治性与复发性，并且有诱发结直肠癌的严重后果。目前，UC疾病治疗以非特异性抗炎治疗为主，特异性差、容易产生抗药性、副作用强，亟需新的治疗方法，而治疗方法的研究离不开可靠的疾病模型。UC传统疾病模型有动物模型与单层细胞培养模型，动物模型与人的发病机制不同且具有异种性，单层细胞模型组成单一且与非恶性细胞之间存在差别，两种模型均存在较为明显的缺点，而UC患者来源的的类器官具有人源性、无恶性特征、包含了复杂的系统并可高通量操作，可以克服传统模型的缺点并集合其优点。目前，国内患者来源的UC结肠类器官相关研究与应用仍然空白，主要面临结肠类器官培养难度高、培养体系不完善以及患者来源样本难以获得的难题。本论文建立了以UC患者为核心的人源结肠类器官样本库，并基于该样本库，对课题组发现的具有UC疾病治疗功能的去抗凝肝素衍生物功效及其机制进行了探究。首先，建立并优化了人源结肠类器官培养的患者-样本-隐窝-类器官系统化流程，流程可行性与可靠性较高。在该流程下，人源结肠类器官能够进行生长发育与分化，生长状态良好，形成完整、清晰的隐窝状结构，复现肠道上皮重要细胞类型；冻存复苏以及传代操作对类器官影响有限。其次，依托北京清华长庚医院消化内科，初步建立了人源结肠类器官样本库，包括UC组、CD组以及正常组。不同疾病来源的结肠类器官差异特征显著，并可以与疾病特征吻合；UC结肠类器官呈现出干性相关细胞的过度的不正常分化，而CD结肠类器官则呈现出干细胞分化能力的显著降低，两类类器官的共同点为杯状细胞的耗竭。最后，基于人源结肠类器官样本库，利用单细胞测序方法，发现去抗凝肝素衍生物LNA与S6对UC结肠类器官生长分化有显著的调控作用，且作用效果并非短期，主要作用机制在于促进祖细胞、干细胞以及TA细胞的正常分化，形成更多的具有功能的上皮细胞、杯状细胞、M细胞，缓解UC疾病中杯状细胞耗竭的情况，同时对上皮屏障起到了修复作用。本论文结合实验室科学与临床医学研究，建立了UC患者疾病模型，发现了去抗凝肝素衍生物促进干性相关细胞分化的UC治疗机制，为病理探究、药物筛选与药物机理研究提供了方法基础。
Ulcerative colitis (UC) is a non-specific inflammatory disease of the rectum and colon, and is a classic inflammatory bowel disease along with Crohn‘s disease (CD).UC is refractory and recurrent, and has the serious consequence of inducing colorectal cancer. Currently, UC disease treatment is based on non-specific anti-inflammatory therapy, which is poor in specificity, easy to produce resistance, and has strong side effects. New therapeutic methods are urgently needed, and the research of therapeutic methods cannot be done without reliable disease models. Traditional disease models of UC are animal models and monolayer cell culture models. Animal models are different from human pathogenesis and are inherently heterogeneous. Monolayer cell models have a single composition and differ from non-malignant cells, so they cannot reflect the epithelial damage in UC. Both traditional models have obvious drawbacks.UC patient-derived organoids as disease models are human-derived, non-malignant, contain complex systems and can be manipulated at high throughput, overcoming the disadvantages and combining the advantages of traditional models.At present, there are still gaps in research and applications related to UC patient-derived colonoid in China, mainly facing the challenges of high difficulty of colonoid culture, imperfect culture system and difficulty in obtaining patient-derived samples.We have established a library of human-derived colonic organoids with UC patients as the core, and based on this library, we have investigated the drug effect and mechanism of the de-anticoagulated heparin derivatives with UC disease therapeutic function.First, a systematic patient-sample-crypt-organoid process for human-derived colonic organoid culture was established and optimized, which is feasible and reliable. Under this process, patient-derived colonoids are able to grow and differentiate well, forming complete and clear crypt-like structures, recreating important cell types of colon epithelium. Also, resuscitation operations and passaging operations had limited effects on colonoids. Secondly, relying on the Department of Gastroenterology of Tsinghua Chang Gung Hospital in Beijing, we initially established a library of patient-derived colonoids, including UC group, CD group and wild type group. The colonoids from different disease sources had significant differential characteristics and could be matched with disease characteristics.UC colonoids show excessive abnormal differentiation of stemness-associated cells, while CD colonoids show a significant reduction of stem cell differentiation capacity, and both of them show the depletion of globet cells.. Finally, based on the library, using the single-cell sequencing method, we found that LNA and S6, derivatives of de-anticoagulated heparin, have significant regulatory effects on the growth and differentiation of UC colonoids, and the effects were not short-term. The main mechanism is to promote the normal differentiation of progenitor cells, stem cells, and TA cells to form more functional epithelial cells, globet cells, and M cells, alleviating the depletion of globet cells in UC disease, while providing a repairing effect on the epithelial barrier. This thesis combined laboratory science and clinical medicine to establish a novel disease model of UC, and discovered that the de-anticoagulated heparin derivatives promote stemness-related cell differentiation to cure UC, which can provide a platform for pathological investigation, drug screening and drug mechanism research, also making personalized medicine possible.This thesis combined laboratory science and clinical medicine to establish a novel disease model of UC, and discovered that the de-anticoagulated heparin derivatives promote stemness-related cell differentiation to cure UC, which can provide a platform for pathological investigation, drug screening and drug mechanism research of UC treatment.
